Ca2+ ion is a ubiquitous messenger for numerous cellular processes. They are generated by various Ca2+ transporters, delivered globally or locally, and decoded by different effectors according to the signal amplitude and frequency. In pulmonary arterial smooth muscle cells (PASMCs), Ca2+ signals that originate from ryanodine receptors (RyRs) play major roles in agonist- and hypoxia-induced vasoconstriction. Our recent study shows that all three RyR subtypes, RyR1, RyR2 and RyR3, are expressed in PASMCs; and these specific RyR-gated Ca2+ stores are distributed heterogeneously in subsarcolemmal, perinuclear and nuclear sites. Local Ca2+ transients, or Ca2+ sparks generated from these RyR sites are different in their kinetic properties. Since RyR subtypes are dissimilar in their Ca2+ sensitivity for activation and their affinity to various ligands, their heterogeneous distribution raises the intriguing possibility that they may regulate independently different physiological functions. In PASMCs, Ca2+ spark activation causes membrane depolarization, in contrast to hyperpolarization in systemic vascular smooth muscle cells (VSMCs), presumably through activation of Ca2+ activated Cl- channels. This suggests unique interactions with membrane ion channels. RyRs are modulated by multiple mechanisms. Cyclic adenosine diphosphate-ribose (cADPR) is a major endogenous messenger for the RyR-gated Ca2+ release. Studies in VSMCs suggest that vasoactive agonists could mobilize Ca2+ from RyR-gated stores through cADPR. Accumulation of cADPR in pulmonary arteries has also been implicated as the trigger for hypoxic pulmonary vasoconstriction (HPV). RyR activity can also be modulated through interactions with other Ca2+ stores. Recent evidence suggests that the novel nicotinic acid adenine dinucleotide phosphate (NAADP)-sensitive lysosomal Ca2+ store generates localized Ca2+ bursts, which cross-activate RyRs to elicit Ca2+ waves. We have recently identified the lysosomal acidic organelles in PASMCs, and detected a lysosome-dependent Ca2+ release induced by the integrin-specific ligand GRGDSP peptide, suggesting that NAADP-sensitive Ca2+ stores are involved in agonist-specific Ca2+ signaling. Based on this information, we propose that Ca2+ signaling in PASMCs is regulated by the heterogeneous RyR-gated Ca2+ stores; their specific subcellular distributions and interactions with endogenous signaling pathways and Ca2+ compartments allow the differential regulation of cellular functions. To test this hypothesis, we will apply a combination of state-of-the-art techniques including whole-cell patch clamp, laser-scanning confocal microscopy, and UV-pulse laser flash photolysis, siRNA, and CD38 knockout mice to determine (1) the Ca2+ signals and physiological functions mediated by different RyR subtypes; (2) the functional interactions between Ca2+ spark and sarcolemmal ion channels; (3) the physiological functions of the cADPR and ADP-ribosyl cyclase; and (4) the functional interactions of RyR- and NAADP-sensitive Ca2+ stores in PASMCs. This project will provide novel information on the unique control of pulmonary circulation by subcellular local Ca2+ signals. PUBLIC HEALTH RELEVANCE: Ca2+ signaling in pulmonary arterial smooth muscle cell is one of the most important processes in the regulation of pulmonary circulation, and dysfunction of this process leads to severe pulmonary diseases. The proposed project will elucidate at the molecular, cellular and subcellular levels, the physiological functions and regulations of the Ca2+ release channels, namely the ryanodine receptors, as well as their interactions with other ionic channels and intracellular Ca2+ stores (in particular the NAADP-sensitive stores). The information generated by the proposed experiments will be valuable for the development of new treatments for pulmonary vascular diseases.